MDR1 variants and methods for their use

ABSTRACT

This invention provides the identification of a truncation polymorphism of the mdr1 gene that is linked to ivermectin sensitivity in subjects, such as collies. Also provided are methods for detecting drug transport sensitivity in a subject, and animal models and in vitro cell systems using cells from animals having an mdr1 truncation.

REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 10/896,434,filed Jul. 21, 2004 now U.S. Pat. No. 7,393,643, which is a divisionalof U.S. patent application Ser. No. 10/044,671, filed Jan. 10, 2002, nowU.S. Pat. No. 6,790,621, issued Sep. 14, 2004, which claims the benefitof U.S. Provisional Application 60/261,578, filed Jan. 12, 2001, andU.S. Provisional Application 60/314,829, filed Aug. 24, 2001. All ofthese applications are incorporated herein by reference in theirentirety.

FIELD

This disclosure relates to methods and kits for detecting a subject'ssensitivity to pharmaceutical agents, particularly an animal'ssensitivity to application of drugs (such as ivermectin) that interactwith P-glycoprotein. It also relates to variants of the mdr1 gene, whichvariants impact transport of drugs that interact with theP-glycoprotein, as well as cell and whole animal systems comprising suchvariants and methods of using these systems.

BACKGROUND

The observation, over 100 years ago, that certain chemical dyes injectedinto the peripheral circulation were able to gain access to most organsbut not the brain led to the concept of a blood-brain barrier. Researchin the 1960's demonstrated that the anatomical basis of the blood-brainbarrier is the specialized endothelial cells of brain capillaries. Whileit has been thought that the entry of drugs, toxins, and xenobioticsinto the brain is simply a function of lipophilicity, electrical charge,and molecular weight, ongoing research demonstrates that the capillaryendothelium composing the blood-brain barrier is not simply an anatomicentity. A number of active transport systems exist that selectivelyregulate both influx and efflux of compounds across brain capillaryendothelial cells. The most important drug-efflux system of theblood-brain barrier identified to date is P-glycoprotein.

P-glycoprotein, the product of the mdr1 (multidrug resistance) gene, isa 170-kD membrane-spanning, cell-surface protein that functions as adrug-efflux pump. P-glycoprotein was first identified over 20 years agoin chemotherapeutic drug-resistant tumor cells, and is now known to be amajor cause of multidrug resistance in human and veterinary cancerpatients. In tumor cells, P-glycoprotein functions as an ATP-dependentefflux pump resulting in decreased intracellular drug accumulation andreduced cytotoxicity. Chemotherapeutic drugs that are substrates forP-glycoprotein include Vinca alkaloids (vincristine and vinblastine),doxorubicin and related compounds, taxanes, and epipodophyllotoxins.Alkylating agents, platinum compounds, and antimetabolites are notsubstrates for P-glycoprotein. Though these agents are structurally andfunctionally dissimilar, P-glycoprotein substrates share several othercharacteristics. They typically are complex, hydrophobic, amphipathiccompounds that are natural products (i.e., derived from plants ormicro-organisms) or analogs of natural products. A number ofnon-cytotoxic compounds have been identified as P-glycoproteinsubstrates, including steroid hormones, bilirubin, antiparasitic agents,selected antimicrobial agents, and others.

P-glycoprotein is expressed not only in tumor cells, but also in avariety of normal tissues, including renal tubular epithelium,canalicular surfaces of hepatocytes, adrenal cortical cells, colonic andintestinal epithelium, placenta, apical margins of bronchiolarepithelium, and brain capillary endothelial cells. Consistent with itsfunction as a transport pump, the expression of P-glycoprotein innon-neoplastic tissues suggests a normal physiologic role forP-glycoprotein mediating the export of potentially toxic xenobioticsfrom the body. Although the normal function of P-glycoprotein in many ofthese tissues has not been elucidated, a great deal is known about itsrole in the blood-brain barrier.

Avermectins are a class of natural products with broad antiparasiticactivity. Ivermectin, a semi-synthetic lactone in the avermectin family,is a drug that is used extensively in veterinary medicine to treat andcontrol infections caused by nematode and arthropod parasites. It isalso used in human medicine to treat onchocerciasis, lymphaticfilariasis, and strongyloidiasis. Ivermectin induces a tonic paralysisin invertebrate organisms by potentiating glutamate-gated chloridechannels, and/or gamma-amino butyric acid (GABA)-gated chloride channels(Tracy and Webster, In: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9^(th) edition. Hardman et al., eds. New York:McGraw-Hill, 1996: 1009-1026, 1996) of the peripheral nervous system. Inmost mammals, the blood-brain barrier prevents access of ivermectin tothe central nervous system, and since GABA receptors in mammals arerestricted to sites within the central nervous system, mammals aregenerally protected from the neurologic effects of ivermectin (Fisherand Mrozik, Annu. Rev. Pharmacol. Toxicol. 32:537-553, 1992).

There are some specific subgroups of mice and dogs, however, that areexquisitely sensitive to the neurologic actions of ivermectin.Genetically engineered mdr1a knock-out [mdr1a(−/−)] mice are 50 to 100times more sensitive to ivermectin-mediated neurotoxicity than wild-typemice (Schinkel et al., Cell. 77:491-502, 1994), and accumulate80-90-fold higher concentrations of ivermectin in the brain than dowild-type mice. The protein product of mdr1a, called P-glycoprotein(P-gp) is a 170-kD transmembrane protein pump that is present at highconcentrations in the apical membrane of brain capillary endothelialcells (Van Asperen et al., J. Pharmaceut. Sci. 86:881-884, 1997, 1997;Tsuji, Therap. Drug Monitor. 20:588-590, 1998). Substrates of P-gpinclude a variety of large, structurally unrelated hydrophobiccompounds, including naturally occurring compounds such as ivermectin,cyclosporin, digoxin, and others. After substrates are bound by P-gp,they are actively extruded from the endothelial cell into the capillarylumen (Van Asperen et al., J. Pharmaceut. Sci. 86:881-884, 1997).Abrogation of P-gp results in failure of the blood-brain barrier. Highconcentrations of ivermectin accumulate in brain tissue from mdr1a (−/−)mice, and neurotoxicity ensues.

Approximately 25% of a population of the CF-1 mouse strain were muchmore sensitive to neurotoxicity produced by ivermectin than unaffectedmice of the same strain (Umbenhauer et al., Toxicol Appl. Pharmacol.146:88-94, 1997). Investigation into the cause of this sensitivity ledto the discovery that the sensitive animals did not express P-gp intheir brain endothelial cells. Furthermore, arestriction-fragment-length polymorphism in the murine mdr gene wasdocumented that allowed prediction of sensitive animals, and aninheritance pattern following normal Mendelian genetics was observed(Umbenhauer et al., Toxicol Appl. Pharmacol. 146:88-94, 1997).

In dogs, a breed-related sensitivity to ivermectin has been reported inCollies, that may affect 30 to 50% of the population (Pulliam et al.,Veter. Med. 7:33-40, 1985; Hsu et al., Comp. Contin. Educat. Veter.Pract. 11:584-589, 1989, Paul et al., Am. J. Vet. Res. 48:685-688,1987). In one study, 1/200^(th) of the lethal dose of ivermectin forBeagles was lethal for Collies (Pulliam et al., Veter. Med. 7:33-40,1985). Other related canine breeds believed to be affected by ivermectinsensitivity include Border Collies, Shetland Sheepdogs, Old EnglishSheepdogs, and Australian Shepherds (Campbell and Benz, J. Vet.Pharmacol. Therap. 7:1-16, 1984).

Despite numerous investigations (Vaughn, et al., Vet. Res. Commun.13:47-55, 1989; Roher et al., Vet. Res. Commun. 14:157-165, 1990;Pulliam et al., Veter. Med. 7:33-40, 1985), the mechanism forivermectin-sensitivity in Collies is unknown.

SUMMARY OF THE DISCLOSURE

The disclosure provides a mutation in the mdr1 gene, which results inproduction of truncated and non-functional P-gp and thereby causessensitivity to ivermectin and other drugs that serve as P-gp substrates.With the identification of this mutation, methods are provided todetermine if an individual subject is sensitive to ivermectin. Alsoprovided are systems for examining the importance of P-gp in drugtransport in whole animal and cell culture systems.

A provided embodiment is a method of detecting ivermectin sensitivity ina subject (for instance a mammal, such as a canine animal), which methodincludes determining whether a gene-truncation mutation in amdr1-encoding sequence of the subject or a truncated P-pg is present inthe subject. Such a gene-truncation mutation or truncation of P-gpindicates that the subject is sensitive to ivermectin. In specificexamples of such methods, the gene truncation mutation is a deletion ofabout four base pairs at about residue 294-297 of SEQ ID NO: 1 (thecanine mdr1 cDNA) or a homologous cDNA or gene.

In certain embodiments, methods provided herein are used to evaluatewhether the subject can be treated safely with ivermectin or anotherdrug that can be excluded from the brain by P-gp (such as those listedin Table 2).

In certain provided methods, the method includes determining whether thesubject is homozygous or heterozygous for the gene-truncation mutation.

In specific examples of the provided methods, determining whether agene-truncation mutation is present in the subject includes subjectingDNA or RNA from the subject to amplification using oligonucleotideprimers, for instance in performing an oligonucleotide ligation assay.

In a specific embodiment provided herein, the method of detectingivermectin sensitivity in a subject involves obtaining a test sample ofDNA containing a mdr1 sequence of the subject; and determining whetherthe subject has the gene-truncation mutation in the mdr1 sequence,wherein the presence of the mutation indicates sensitivity toivermectin. In certain examples of this embodiment, determining whetherthe subject has the mutation comprises using restriction digestion,probe hybridization, nucleic acid amplification, or nucleotidesequencing.

Further embodiments of methods provided herein involve obtaining fromthe subject a test sample of DNA comprising an mdr1 sequence; contactingthe test sample with at least one nucleic acid probe for an mdr1 genetruncation mutation that is associated with ivermectin sensitivity, toform a hybridization sample; maintaining the hybridization sample underconditions sufficient for specific hybridization of the mdr1 sequencewith the nucleic acid probe; and detecting whether the mdr1 sequencespecifically hybridizes with the nucleic acid probe, wherein specifichybridization of the mdr1 sequence with the nucleic acid probe indicatesivermectin sensitivity. In specific examples of such embodiments, theprobe is present on a substrate, for instance a nucleotide array.

Also provided are methods of detecting ivermectin sensitivity in asubject by determining whether truncated P-gp is present in a samplefrom the subject. Certain examples of such methods will involve reactingat least one P-gp molecule contained in the sample from the subject witha P-gp-specific binding agent (such as an antibody) to form a P-gp:agentcomplex. Such methods can further include detecting the P-gp:agentcomplex, for instance by Western blot assay, ELISA, or other immunoassaytechnique.

Also provided herein are kits for use in diagnosing ivermectinsensitivity in a subject. Such kits include at least one probe thatspecifically hybridizes to an mdr1 gene-truncation mutation associatedwith ivermectin sensitivity. In specific examples of such kits, theprobe specifically hybridizes to an mdr1 gene-truncation mutation at orabout residue 294-297 of SEQ ID NO: 1.

Other provided kits for use in diagnosing ivermectin sensitivity in asubject contain a P-gp-specific binding agent, such as an antibody. Inspecific examples of such kits, the provided agent is capable ofspecifically binding to truncated P-gp protein.

Also provided herein are oligonucleotides that specifically hybridize toa canine mdr1 gene-truncation mutation, for instance an oligonucleotidethat hybridizes to an mdr1 gene-truncation mutation at residue 294-297.

Other embodiments are systems and methods for studying the effects ofdrugs (and drug candidates) on biological systems expressing a mdr1 genetruncation, such as the mdr1 gene-truncation mutation at residues294-297. Examples of such systems include cultured cells (such asintestinal epithelial cells, brain endothelial cells (for instance,capillary endothelial cells), renal-tubular cells, hepatocytes, orneoplastic cells) isolated from a canine that naturally exhibits a genetruncation mutation in the mdr1 gene. Other examples of such systemsinclude animal models (including for instance dogs) in which the mdr1gene is naturally truncated, or in which such truncations have beenengineered using recombinant technologies and/or cloning. Methods arealso provided for using these animal models and cell systems, forinstance to study drug interactions with P-gp or to examine the impactof drugs and drug candidates on biological systems. Such methods wouldbe particularly useful in the drug approval process.

One embodiment is a method of determining a P-gp influenced biologicaleffect of a compound on a canine cellular system, which method involvescontacting a canine cell having a truncation mutation in its mdr1 genewith the compound, and comparing a characteristic (such as a genetic,physiological, chemical, or morphological characteristic) of the caninecell contacted with the compound with the characteristic of a similarcanine cell that was not contacted with the compound. In such methods, adifference in the characteristic between the two cells is indicative ofthe P-gp influenced biological effect in the cell. In specific examplesof such methods, the canine cell is a Collie cell. The truncationmutation in the mdr1 gene is in some examples a mutation at residue294-297.

Specific types of canine cells include, but are not limited to,gastrointestinal tissue cells, renal tissue cells, nerve tissue cells,brain capillary endothelial cells, and liver tissue cells. In somemethods, the canine cell is a neoplastic cell.

Also provided are methods of determining a P-gp influenced biologicaleffect of a compound on a canine cellular system, wherein contacting thecanine cell with the compound occurs in vivo in the native environmentof the canine cell, for instance in a dog (such as a Collie dog).

In some examples of the provided methods, biological effects includeabsorption or distribution of a drug or compound, for instance a drug orcompound that interacts with or is transported by P-gp.

Also provided is an animal model useful for studying a P-gp influencedbiological effect of a compound, comprising a Collie identified as beinghomozygous or heterozygous for a truncation mutation in the mdr1 gene(for instance, a mutation at residue 294-297). Also provided are methodsof using this animal model to examine the effect of compounds thatinteract with P-gp, for instance compounds that are transported by P-gpor that modulate its transport activity.

Compounds contemplated for use in the methods provided herein includeanti-infective agents (e.g., antiviral, antibacterial, or anti-prionagents), antineoplastic agents, analgesics, neurokinin receptorantagonists, anti-emetic agents, beta-adrenergic receptor antagonists,antiepileptic agents, anti-psychotic agents, anti-depressive agents, andother drugs that act on the central nervous system.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a semi-quantitative reverse transcriptase PCR analysis ofmdr1 in blood samples from ivermectin-sensitive (samples 15 and 16) andnon-sensitive (resistant) (samples 17 and 18) Collies. Duplicatereactions were assayed for each dog as shown using canine mdr1 and GAPDHprimers. The resultant amplicons were separated by electrophoresisthrough an agarose gel. The arrows indicate the expected size of themdr1 (P-gp) and GAPDH products.

FIG. 1B is a graph, quantitating the UV fluorescence of mdr1 and GAPDHreverse transcriptase PCR products obtained by digital image analysis ofthe ethidium bromide-stained gel shown in FIG. 1A.

FIG. 2 is a sequence comparison of wild-type (nucleotides 275 to 708 ofSEQ ID NO: 1) (top) and mutant (nucleotides 275 to 293 and 298 to 708 ofSEQ ID NO: 1) (bottom) mdr1 cDNAs. As demonstrated herein, a four basepair deletion is present in the mutant cDNA. Codons in the vicinity ofthe deletion are indicated by brackets for both the wildtype and mutantcDNAs. Bolded letters indicate stop codons created in the mutant cDNA asa result of the frame-shift. The dashed box indicates the palindromicsequence in the vicinity of the deletion mutation.

FIG. 3 is a diagrammatic representation of the transmembrane structureof P-gp (Gottesman and Pastan, Annu. Rev. Biochem. 62:385-427, 1993).The mutation site (arrowhead) occurs at amino acid 75, resulting in aframe shift that generates several downstream stop codons, the first twoof which occur at amino acid positions 91 and 111. Greater than 90% ofthe protein is predicted to be missing in dogs homozygous for the mutantallele due to the truncation.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three-letter code for amino acids, as defined in 37 C.F.R.§1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 shows the nucleotide sequence of the canine mdr1 cDNA(GenBank Accession No. AF045016), and the P-gp amino acid sequenceencoded thereby.

SEQ ID NO: 2 shows the amino acid sequence of canine P-gp.

SEQ ID NOs: 3-10 show respective synthetic oligonucleotides used toprimer in vitro amplification reactions of the canine mdr1 gene, asdescribed in Example 1.

DETAILED DESCRIPTION

I. Abbreviations

mdr multidrug resistance gene

P-gp P-glycoprotein, protein product of the mdr1 gene

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 2000 (ISBN 0-19-879276-X); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise explained herein, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. The singularterms “a,” “an,” and “the” include plural referents unless contextclearly indicates otherwise. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingexplanations of terms, will control. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting.

III. Identification of Truncation Mutations in mdr1, Responsible forCollie Sensitivity to Ivermectin.

There is a sub-population of Collies and dogs of related breeds thatdisplay a similar sensitivity to ivermectin. The inventors havesurprisingly discovered that a deletion mutation of the mdr1 gene existsin ivermectin-sensitive Collies. The mutation produces a frame shiftthat generates a premature stop codon in the mdr1 gene resulting in aseverely truncated, nonfunctional protein. Collies homozygous for thedeletion (mutant/mutant) exhibit ivermectin sensitivity while those thatare heterozygous (normal/mutant) or homozygous normal are not sensitiveto ivermectin neurotoxicity. Several other breeds, including Australianshepherds, Shelties, and Old English Sheepdogs have been reported toexhibit ivermectin sensitivity; it is believed that susceptibleindividuals of these species may also display truncation mutations inthe mdr1 gene.

The identification of the naturally occurring mdr1 truncation mutationsin dogs has enabled the use of animals carrying such a mutation, and ofcells derived from such animals, to study interactions of drugs (orpotential drugs) with P-gp and the systemic effects of suchinteractions. These animal and cell-based systems are particularlyuseful for identifying and characterizing ways to:

(a) improve, regulate, or prevent gastrointestinal absorption of drugs;

(b) improve, regulate, or prevent brain penetration of drugs (e.g.,increasing brain penetration of HIV-1 drugs; drugs for treating priondiseases; antineoplastic agents; or compounds for pain or depression);

(c) improve, regulate, or prevent renal excretion of drugs;

(d) improve penetration of drugs into tumor cells;

(e) improve, regulate, or prevent biliary excretion of drugs; and

(f) improve, regulate or prevent penetration of drugs through theplacenta.

These systems are also particularly useful to study the effects offunctional mdr1 polymorphism(s), particularly in order to understand theeffects of such polymorphisms as they may be found in additionalspecies, including for instance humans. The systems can also be used tostudy the effects (drug interactions) that a highly effective, potentP-gp inhibitor would have in an intact biological system, and thusprovides an excellent method for examining the large-mammal effects ofdrugs, for example during the drug approval and testing process.

The invention is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 Identification of a Truncation Mutation in Colliemdr1 Materials and Methods

General Procedures and Materials

Procedures used in research disclosed herein were approved by theInstitutional Animal Care and Use Committee.

Blood samples (8 ml) were obtained by jugular venipuncture from each of13 clinically healthy Collies (Wil-O-Lane Kennels, Allegan, Mich.). AllCollies used in the study had previously been identified asivermectin-sensitive or -nonsensitive by observing dogs for signs ofneurotoxicosis after oral administration of 120 μg of ivermectin per kgof body weight (20 times the label dose for heartworm prevention), aspreviously described (Paul et al., Amer. J. Vet. Res. 61:482-483, 2000;Fassler et al., J. Am. Vet. Med. Assoc. 199:457-460, 1991). Each dog wasidentified by an ear tattoo and housed in a run measuring 4×8 feet withmetal walls and a raised, coated, metal screen floor. Facilitiesexceeded the minimal requirement specified by USDA guidelines.

Seven Collies were identified as ivermectin-sensitive (4 male and 3female) and 6 were not ivermectin-sensitive (3 male and 3 female). Twoof the Collies in the study were littermates and two others werenon-littermate siblings. Of the two littermates, one was sensitive toivermectin, the other was not. Both of the non-littermate siblings weresensitive to ivermectin. Several other Collies in this study sharedeither the same sire or the same dam. Two of the common sires and threeof the common dams were represented by offspring in both theivermectin-sensitive and -nonsensitive groups. Additional blood sampleswere obtained from 4 non-Collie dogs, including 1 Beagle, 2 GoldenRetrievers, and 1 Staffordshire terrier cross-bred dog.

Semiquantitative RT-PCR of Canine mdr1 Gene

Total RNA was extracted from venous blood leukocytes using TRIzolreagent (Gibco BRL). Blood leukocytes were prepared by density gradientcentrifugation. For RT reactions, a GeneAmp RT PCR kit (Perkin-Elmer)was used with oligo(dT) primers. Equivalent amounts of cDNA were thenamplified in separate PCR reactions using Amplitaq (Perkin Elmer) with2.5 mM MgCl₂.

PCR consisted of either 20 (GAPDH) or 27 (mdr1 product A) cycles, withdenaturing, annealing, and extension conditions of 95° C. (15 seconds),60° C. (15 seconds), and 72° C. (60 seconds) in a Perkin-Elmerthermocycler (2400 GeneAmp PCR System). The number of cycles for eachproduct was determined on the basis of kinetic studies to ensure thatthe amplification reaction was within the logarithmic (not plateau)range. PCR products were resolved by electrophoresis in 1% agarose gelscontaining ethidium bromide. Expected sizes of the GAPDH and mdr1 A bandare 229 bp and 892 bp, respectively. The UV fluorescence of DNA bandswas measured with an IS-1000 Digital Imaging System (Alpha Innotec). Forindividual dogs, the fluorescence value for the mdr1 A product isdivided by the fluorescence value of the GAPDH product to allow directcomparison between dogs.

Sequencing of Canine mdr1 cDNA

Four primer pairs, amplifying four products (referred to herein as A, B,C, and D) spanning 95% of the canine mdr1 cDNA (GenBank Accession No.AF04016) were designed for sequencing experiments (see, Table 1). UsingRNA from three of the sensitive Collies, cDNA was synthesized in RTreactions as described above and amplified in separate reactions. Forproducts A, B, and C, PCR was accomplished using the conditionsdescribed above, using 35 cycles. PCR for product D consisted of 35cycles, with denaturing, annealing, and extension conditions of 95° C.(10 seconds), 64.5° C. (15 seconds), and 72° C. (150 seconds). OptimalMgCl₂ concentrations were 2.5 mM for PCR products A, C, and D, and 1.5mM for PCR product B. For initial experiments, products generated fromsamples of three different dogs were ligated into pGEM-T Easy (ProMega),which was then used to transform ElectroMAX DH10B E. coli cells byelectroporation (Gene Pulser II, BioRad). Plasmid DNA was isolated(Plasmid Mini Kit, Qiagen) and sequenced by Davis Sequencing Inc.(Davis, Calif.) using dye-terminator chemistry and an automated DNAsequencer (ABI 377, PE Applied Biosystems). For all subsequentexperiments, sequencing of PCR products (Davis Sequencing Inc.)following purification (Qiaquick PCR Purification Kit, Qiagen) wasperformed. Sequences from experimental dogs were compared to the knowncanine mdr1 sequence (GenBank AF 045016; SEQ ID NO: 1).

TABLE 1 Oligonucleotide primers used in this study. The combination ofprimer pairs used in this study provides >95% coverage of the caninemdrl cDNA. Mdrl pro- duct Size de- of sig- PCR na- Posi- Pro- tionPrimer tion duct A Forward: 2942- 892 5′- TCC GGT TTG GTG CCT ACT TG¹2961 Reverse: 3833- 5′- TGC TCC TTG ACT TTG CCA TTC² 3814 B Forward:2421- 1021 5′- CCT CAC TAA GCG GCT TCG ATA C³ 2441 Reverse: 3441- 5′-AAA CAG GAT GGG CTC CTG AGA C⁴ 3420 C Forward:  168- 1061 5′- CAG CACGTT TGC AAT GTT TC⁵  189 Reverse: 1228- 5′- TCT GGT TTA TGT CCA CTC TTCG⁶ 1208 D Forward: 1112- 1432 5′- AGG CAT CCC CAA GCA TTG AAG⁷ 1132Reverse: 2543- 5′- TGA GCC GCA TCA TTG GCA AG⁸ 2524 ¹Corresponds to SEQID NO: 3. ²Corresponds to SEQ ID NO: 4. ³Corresponds to SEQ ID NO: 5.⁴Corresponds to SEQ ID NO: 6. ⁵Corresponds to SEQ ID NO: 7. ⁶Correspondsto SEQ ID NO: 8. ⁷Corresponds to SEQ ID NO: 9. ⁸Corresponds to SEQ IDNO: 10.

Results

We took advantage of a well-defined population of ivermectin-sensitiveand non-sensitive Collies (Paul et al., Amer. J. Vet. Res. 61:482-483,2000: Fassler et al., J. Am. Vet. Med. Assoc. 199:457-460, 1991).Sensitive animals were designated as those that experienced clinicalsigns of neurologic toxicity after receiving a single, oral dose ofivermectin (120 μg/kg). Clinical signs of neurotoxicity that wereevaluated include apparent depression, ataxia, mydriasis, salivation, ordrooling (Paul et al., Amer. J. Vet. Res. 61:482-483, 2000).Semi-quantitative reverse transcriptase PCR analysis was conducted onRNA isolated from ivermectin-sensitive and non-sensitive Collies todetermine if mdr1 expression is lower in sensitive Collies than innon-sensitive Collies. Amplification of a 229-bp product of the canineglyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as aninternal standard to control for variability in reverse transcriptaseand PCR reactions. FIG. 1A shows representative ethidium bromide-stainedRT-PCR products of GAPDH (229 bp) and mdr1 (product A, 892 bp) fromivermectin sensitive (samples 15 and 16) and nonsensitive (samples 17and 18) Collies. Individual UV fluorescence intensity values for GAPDHand mdr1 cDNA derived from these gels are shown in FIG. 1B. The level ofmdr1 gene expression, as determined by semiquantitative reversetranscriptase PCR, did not differ between ivermectin-sensitive andnon-sensitive Collies.

A reverse transcriptase PCR strategy was used to clone mdr1 cDNA fromone normal dog (beagle) and 3 ivermectin-sensitive Collies. Sequencedata from the beagle mdr1 cDNA was identical to that reported for normalcanine mdr1 cDNA (GenBank Accession No. AF045016). Sequence analysis ofclones from the three ivermectin-sensitive Collies revealed an identicalfour base pair deletion within the first 10% of the transcript (FIG. 2).This deletion causes a frame-shift mutation at nucleotide 294 of thetranscript, which corresponds to amino acid 75 (FIG. 3). The frame shiftgenerates several stop codons (FIG. 3), the first of which occurs atamino acid position 91 of P-gp (GenBank Accession No. AF04016; SEQ IDNO: 2). The result is a severely truncated, nonfunctional, protein.

For all subsequent experiments, a PCR-based strategy was used forsequencing mdr1 cDNA so that heterozygosity could be readily detected.The exact four-nucleotide deletion (AGAT) was detected in all (7/7)samples from ivermectin-sensitive Collies. Furthermore, these dogs werehomozygous for the deletion. Samples from all non-Collie dogs (1 Beagle,2 Golden Retrievers, and 1 Staffordshire terrier cross-bred dog) werehomozygous wild-type. Interestingly, samples from all (6/6) ivermectinnon-sensitive Collies displayed a heterozygous genotype, with one strandcarrying the mutant allele, and the other strand carrying the wild-typeallele. It is highly unlikely that these findings are a result of chance(Fisher's Exact test, P=0.006).

Discussion

The reported data demonstrate that a frame-shift deletion of four basepairs at the 5′ end of the canine mdr1 gene is associated with (andlikely causes) ivermectin-sensitivity in Collies. Premature terminationof P-gp synthesis as a result of the frame-shift yields a severelytruncated protein that is less than one-tenth its normal size, based onthe predicted amino acid sequence. P-glycoprotein's drug-efflux functionis dependent upon ATP binding sites, substrate binding sites,phosphorylation sites, and multiple membrane-spanning motifs (Yoshimuraet al., J. Biol. Chem. 264:16282-16291, 1989; Skach and Lingappa, CancerRes. 54:3202-3209, 1994). Since none of these required elements arepresent in the truncated protein, we conclude that animals homozygousfor the deletion do not express a functional form of P-gp.

The pivotal role that P-gp plays in protecting the brain from ivermectinhas previously been established (Kwei et al., Drug Metab. Dispos.27:581-587, 1999; Marques-Santos et al., Pharmacol. Toxicol. 84:125-129,1999; Schinkel et al., J. Clin. Invest. 97:2517-2524, 1996; Van Asperenet al., J. Pharmaceut. Sci. 86:881-884, 1997). Because a large number ofother drugs serve as P-gp substrates, it is possible that affectedCollies would also experience greater sensitivity to the neurologiceffects induced by drugs other than ivermectin. Examples of P-gpsubstrates that might induce neurotoxicity include those listed in Table2 (Tsuji, Therap. Drug Monitor. 20:588-590, 1998; Schinkel et al., J.Clin. Invest. 97:2517-2524, 1996).

TABLE 2 Selected Clinically Relevant Substrates of P-glycoproteinAntineoplastic Antimicrobials and agents Antiparasitics MiscellaneousVincristine* Cefoperazone Digoxin* Vinblastine* TetracylinesCyclosporine A Doxorubicin* Ivermectin* Verapamil* MitoxantroneLoperamide* Paclitaxel* Dexamethasone Ondansetron* *Drugs that havepotential for neurotoxicity

In mice, people, and dogs, P-gp is normally expressed in other tissuesof the body in addition to brain capillaries. Consistent with itsfunction as a transport pump, expression of P-gp occurs at sites whereit might protect the animal from xenobiotics. For example, P-gp isexpressed at high levels in renal proximal tubular cells, liver, smallbowel, colon, and placenta (as well as brain endothelium) (Lum et al.,Pharmacother. 13:88-109, 1993; Ginn, Vet. Pathol. 33:533-541, 1993). Thehighest levels of P-gp expression occur in tumor cells, where P-gpfunctions as a multidrug transporter, protecting tumor cells from avariety of chemotherapeutic drugs including anthracyclines, Vincaalkaloids, taxanes, and epipodophyllotoxins. It is believed thativermectin-sensitive Collies would be less likely to develop multidrugresistant tumors than the general canine population, and would thereforehave better chemotherapy response rates.

The specific cause of the identified mdr1 mutation is unknown. However,it has been reported that unusual DNA structures, including palindromicDNA, promote genetic instability (Lewis et al, Ann. NY Acad. Sci.870:45-57, 1999). Unusual DNA structures are thought to cause DNApolymerase to pause and, consequently, can disrupt DNA replication.Mutational events are not limited to sequences located within thepalindromic DNA, but can also occur in sequences in the vicinity of apalindrome. Therefore, it is interesting to note that a palindromicsequence (GGTTTTTGG (nucleotides 276-284 of SEQ ID NO: 1); FIG. 2)occurs nine bases upstream of the deletion site. Whether or not thispalindromic sequence played a role in generating the four-base pairdeletion in these Collies is unknown.

The inheritance pattern of ivermectin sensitivity in Collies is unknown.Results of the research described here are consistent with an autosomalrecessive inheritance pattern, since only Collies that had two mutantalleles displayed the ivermectin-sensitive phenotype. However, a largersample size will be needed to definitively determine the inheritancepattern. With many genetic diseases in people, affected individuals havemany different mutations throughout the affected gene. However, in thisstudy, all Collies had the same mutation. Because many of these animalswere unrelated, it seems likely that the mutant alleles in these dogshave a common, yet-to-be-determined origin.

Example 2 Frequency of the Mutant MDR1 Allele

This Example provides methods and results from a study to determine thefrequency of the mutant MDR1 allele associated with ivermectinsensitivity in a sample of Collies living in Washington and Idaho. Bloodwas collected from 40 Collies for RNA extraction. The RNA was reversetranscribed, and PCR performed to amplify a 1061-base pair ampliconcontaining the MDR1 mutation. Sequence analysis was performed todetermine the genotype of each dog. Twenty-two percent of the Colliesstudied were homozygous for the normal allele (normal), 42% wereheterozygous (carrier), and 35% were homozygous for the mutant allele(affected).

Materials and Methods

Animals—Forty clinically healthy, client-owned Collies were studied.Owner consent was obtained, and the study was approved by theInstitutional Animal Care and Use Committee. Both rough-coated andsmooth-coated Collies were represented. Dogs included in the samplepopulation were those animals for which the owner was interested indetermining MDR1 genotype in their pet(s). Advertising for Colliesoccurred primarily by announcements at the Inland Northwest Collie Clubmeetings and word-of-mouth. A pedigree (representing the last 4generations) was available for eight animals.

Collection and Extraction of RNA—An 8 ml blood sample was collected fromeach dog for RNA isolation. Blood leukocytes were prepared by densitygradient centrifugation. Total RNA was extracted from venous bloodleukocytes using TRIzol reagent (Gibco BRL).

Reverse transcriptase PCR and sequencing—For reverse transcriptasereactions, a GeneAmp RT PCR kit (Perkin-Elmer) was used with oligo(dT)primers. The cDNA was then amplified (using primers as described inExample 1) in separate PCR reactions using Amplitaq (Perkin Elmer) with2.5 mM MgCl₂. PCR consisted of 36 cycles, with denaturing, annealing,and extension conditions of 95° C. (10 seconds), 64° C. (15 seconds),and 72° C. (60 seconds) in a MJ Research thermocycler (PTC-200). PCRproducts were resolved by electrophoresis in 1% agarose gels containingethidium bromide. Expected size of the MDR1 band was 1061 bp. PCRproducts were purified (Qiaquick PCR Purification Kit, Qiagen) andsequenced by Davis Sequencing Inc. (Davis, Calif.) using dye-terminatorchemistry and an automated DNA sequencer (ABI 377, PE AppliedBiosystems). Sequences from experimental dogs were compared to the knowncanine MDR1 sequence (GenBank AF 045016).

Results

The deletion mutation associated with ivermectin sensitivity in Collieswas present in a large number of dogs in this study. Nine dogs (22%)were homozygous for the normal (wild-type) MDR1 sequence, 14 dogs (35%)were homozygous for the mutant allele, and 17 (42%) were heterozygous.

Analysis of the 8 pedigrees was performed and showed that four of thedogs in the study were closely related. For one pair of siblings, testresults indicated that one dog was affected (homozygous mutant) and theother was heterozygous (one normal allele and one mutant allele). Foranother closely related pair of dogs, a dam and her daughter, testresults indicated that the dam was heterozygous, and the daughter onedog was affected. However, there were two affected dogs that were notrelated to other dogs in the study within the four most recentgenerations. Furthermore, these eight dogs were unrelated (within thefour most recent generations) to a sample population of Collies fromMichigan that were studied previously, in which all animals were eitherheterozygous or homozygous for the described MDR1 mutation.

Discussion

Ivermectin sensitivity in Collies has recently been associated withhomozygous expression of a deletion mutation of the MDR1 gene.P-glycoprotein, the product of the MDR1 gene, is an integral componentof the blood-brain barrier. At the blood brain barrier, P-glycoproteinactively extrudes drugs from brain tissue back into capillaries,resulting in lower brain concentrations of drugs that are substrates forP-glycoprotein (Fromm, Int J Clin Pharm Therap 38:69-74, 2000; Kim etal., J Clin Invest 101:289-294, 1998; Jonker et al., Br J Pharmacol127:43-50, 1999; Schinkel, Int J Clin Pharmacol Ther 36:9-13, 1998). InMDR1 knockout mice, lack of P-glycoprotein leads to abnormally increasedaccumulation of certain drugs in the brain with resultant undesiredneurologic adverse effects (Schinkel, Int J Clin Pharmacol Ther 36:9-13,1998). In ivermectin-sensitive Collies, this mutation consists of a4-base-pair deletion that generates a premature stop codon, resulting ina severely truncated, nonfunctional protein product.

Previous investigators have estimated that up to 30-40% of Collies aresensitive to ivermectin (Pulliam et al., Vet Med 80:33-40, 1985; Paul etal., Am J Vet Res 48:684-685, 1987; Rohrer and Evans, Vet Res Commun14:156-165, 1990). Our study yielded similar results. In our studypopulation, the frequency of the homozygous mutant genotype was 35%.Interestingly, in a separate sample of Collies from Michigan, all dogscarried at least one mutant allele. From the pedigrees available, 8/40from this sample and 15/15 from the Michigan sample, none of theWashington/Idaho dogs were related to the Michigan dogs within the fourmost recent generations. Collectively, these results suggest that theMDR1 mutation associated with ivermectin sensitivity is widely dispersedin the Collie population.

Sporadic descriptions of ivermectin sensitivity have been reported in afew other breeds including Shetland sheepdogs, Australian shepherds, andOld English sheepdogs (Hadrick et al., JAVMA 206:1147-1150, 1995;Paradis, Compend Cont Ed Pract Vet 20:193-200, 1998; Hsu et al., CompeedCont Ed Pract Vet 11:584-588, 1989). Whether or not these breeds sharethe same MDR1 mutation as Collies is unknown. In people, severaldifferent MDR1 mutations have been described, so it is reasonable toassume that other breeds may not share the same MDR1 genotype as doCollies (Cascorbi et al., Clin Pharmacol Ther 69:169-174, 2001; Kerb etal., Pharmacogenomics 1:51-64, 2001).

Determination of the genotype of Collies is important clinically forseveral reasons. First, ivermectin is not the only clinically relevantsubstrate for P-gp that can cause neurotoxicity. The over-the-counterantidiarrheal agent loperamide has been reported to cause neurotoxicityin Collies at doses routinely used in other breeds (Hugnet et al., VetHum Toxicol 38:31-33, 1996). Loperamide, like ivermectin, is generallyexcluded from entering brain tissue in high concentrations by theactions of P-gp. In affected Collies, loperamide achieves highconcentrations in brain tissue causing neurologic toxicity. In supportof this fact, one of the Collies in the present study was treated withan appropriate dose of loperamide as a puppy and developed severe(nearly fatal) neurologic toxicity. The dog tested homozygous for themutant allele. Other drugs that are substrates for P-gp and that cancause neurotoxicity in affected Collies include vincristine,vinblastine, ondansetron, and potentially moxidectin.

There are other, non-neurologic, implications for Collies with the MDR1mutation described. P-glycoprotein is normally also expressed at theluminal border of the intestinal tract (Liu and Hu, Clin Chem Lab Med38:877-881, 2000), where it functions as an “anti-absorption” mechanismfor a number of drugs, including digoxin, cyclosporin A, dexamethasone,antiviral drugs and others (Wacher et al., Advanced Drug Delivery Rev46:89-102, 2001). In affected Collies, oral bioavailability of thesedrugs is likely to be greater than in unaffected dogs. This would resultin higher plasma concentrations and a higher likelihood of adverse drugreactions in affected Collies.

It is likely that a high percentage of Collies presented toveterinarians for treatment are affected by the MDR1 mutation describedin this report. It is important that veterinarians consider this factorwhen selecting pharmacological therapy for Collies. Furthermore, anadverse drug reaction involving neurologic toxicity should be consideredfor Collies exhibiting abnormal CNS signs.

Example 3 Other mdr1 Truncations

With the provision herein of the correlation between a canine mdr1 genetruncation and ivermectin sensitivity, the isolation and identificationof additional mdr1 truncations, and similar truncations in other caninespecies, is enabled. Conventional methods for the identification ofgenetic polymorphisms in a population can be used to identify suchadditional polymorphisms.

For instance, existing populations (e.g., Collie or other populations)are assessed for ivermectin sensitivity (or sensitivity to other drugsthat interact with P-gp), and a subset of individuals within thepopulation (such as those subjects known to be prone to neurotoxicosis,or related individuals) are genotyped as relates to an mdr1 sequence.These mdr1 sequences are then compared to a reference mdr1 sequence,such as the frame-shift truncation allele described herein, to determinethe presence of one or more variant nucleotide positions. After variantnucleotides are identified, statistical analysis of the population canbe used to determine whether these variants are correlated withsensitivity to ivermectin or other drug treatment.

Alternatively, the P-gp protein itself can be analyzed in such subjects,to determine the presence and/or level and/or size of the protein.

Example 4 Clinical Uses of mdr1 Polymorphisms

To perform a diagnostic test for the presence or absence of a truncationmutation (e.g., a deletion, frameshift, point mutation, or other changethat results in a truncated protein product) in an mdr1 sequence of anindividual, a suitable genomic DNA-containing sample from a subject isobtained and the DNA extracted using conventional techniques. Mosttypically, a blood sample, a buccal swab, a hair follicle preparation,or a nasal aspirate is used as a source of cells to provide the DNAsample. The extracted DNA is then subjected to amplification, forexample according to standard procedures. The allele of the truncationmutation is determined by conventional methods including manual andautomated fluorescent DNA sequencing, primer extension methods(Nikiforov, et al., Nucl. Acids Res. 22:4167-4175, 1994),oligonucleotide ligation assay (OLA) (Nickerson et al., Proc. Natl.Acad. Sci. USA 87:8923-8927, 1990), allele-specific PCR methods (Rust etal., Nucl. Acids Res. 6:3623-3629, 1993), RNase mismatch cleavage,single-strand conformation polymorphism (SSCP), denaturing gradient gelelectrophoresis (DGGE), Taq-Man, oligonucleotide hybridization, and thelike. Also, see the following U.S. patents for descriptions of methodsor applications of polymorphism analysis to disease prediction and/ordiagnosis: U.S. Pat. Nos. 4,666,828; 4,801,531; 5,110,920; 5,268,267;and 5,387,506.

The markers of ivermectin sensitivity disclosed herein can be utilizedfor the detection of, and differentiation of, individuals who arehomozygous and heterozygous for the truncation mutation polymorphism(s).The value of identifying individuals who carry a sensitive allele ofmdr1 (i.e., individuals who are heterozygous or homozygous for an allelethat contains a “sensitive” mdr1 polymorphism, such as the truncationdescribed herein) is that therapy for these individuals can then beinitiated or customized (e.g., through avoiding ivermectin or otherdrugs usually kept from crossing the blood-brain barrier by P-gp) toreduce the occurrence of neurotoxicity in these individuals. Likewise,the presence of the allele will assist breeders in breeding programs,either to avoid introducing a sensitive allele into a breedingpopulation, or by selectively avoiding animals carrying such an allele.Information regarding an animal's mdr1 allele status (sensitive,resistant, or heterozygous) could for instance be tested early in thelife of the individual, and included on a license, medical record,pedigree, and so forth.

Example 5 Polymorphism Gene Probes and Markers

Sequences surrounding and overlapping truncation polymorphisms in themdr1 gene can be useful for a number of gene mapping, targeting, anddetection procedures. For example, genetic probes can be readilyprepared for hybridization and detection of the described truncationpolymorphism. Such probe sequences may be greater than about 12 or moreoligonucleotides in length and possess sufficient complementarity todistinguish between a wild-type sequence and a sequence in which fournucleotides (e.g., nucleotides corresponding to positions 294-297 of SEQID NO: 1) have been lost.

Similarly, sequences surrounding and overlapping the specificallydisclosed truncation polymorphism (or other polymorphisms found inaccordance with the present teachings) can be utilized inallele-specific hybridization procedures. A similar approach can beadopted to detect other mdr1 polymorphisms, such as truncations.

Sequence surrounding and overlapping a mdr1 polymorphism, or any portionor subset thereof that allows one to identify the polymorphism, arehighly useful. Thus, another embodiment provides a genetic markerpredictive of the herein-disclosed frame-shift truncation polymorphismof mdr1, comprising a partial sequence of the canine genome including atleast about 10 contiguous nucleotide residues including residues 294-297of SEQ ID NO: 1, or specifically not including these four residues butstill including the surrounding residues (in other words, specific for adeletion mutant in these four residues). Examples of sucholigonucleotides include the following nucleotide sequence:AAACATGACAGATAGCTTTGCAAAT (corresponding to residues 284-309 of SEQ IDNO: 1), and sequences complementary therewith, wherein the underlinedfour nucleotides can be left out to create an oligonucleotide specificfor the disclosed gene truncation mutation.

Example 6 Detecting mdr1 Mutations

The truncation mutation at nucleotide residue 294-297 of SEQ ID NO: 1 ofcanine mdr1 (the first position of which encodes amino acid residue 75of P-gp, SEQ ID NO: 2) can be detected by a variety of techniques. Thesetechniques include allele-specific oligonucleotide hybridization (ASOH)(Stoneking et al., Am. J. Hum. Genet. 48:370-382, 1991), which involveshybridization of oligonucleotide probes to the sequence, stringentwashing, and signal detection. Other applicable methods includetechniques that incorporate more robust scoring of hybridization.Examples of these procedures include the ligation chain reaction (ASOHplus selective ligation and amplification), as disclosed in Wu andWallace (Genomics 4:560-569, 1989); mini-sequencing (ASOH plus a singlebase extension) as discussed in Syvanen (Meth. Mol. Biol. 98:291-298,1998); and the use of DNA chips (miniaturized ASOH with multipleoligonucleotide arrays) as disclosed in Lipshutz et al. (BioTechniques19:442-447, 1995). Alternatively, ASOH with single- or dual-labeledprobes can be merged with PCR, as in the 5′-exonuclease assay (Heid etal., Genome Res. 6:986-994, 1996), or with molecular beacons (as inTyagi and Kramer, Nat. Biotechnol. 14:303-308, 1996).

Another technique is dynamic allele-specific hybridization (DASH), whichinvolves dynamic heating and coincident monitoring of DNA denaturation,as disclosed by Howell et al. (Nat. Biotech. 17:87-88, 1999). A targetsequence is amplified (e.g., by PCR) using one biotinylated primer. Thebiotinylated product strand is bound to a streptavidin-coated microtiterplate well (or other suitable surface), and the non-biotinylated strandis rinsed away with alkali wash solution. An oligonucleotide probe,specific for one allele (e.g., the wild-type allele), is hybridized tothe target at low temperature. This probe forms a duplex DNA region thatinteracts with a double strand-specific intercalating dye. Whensubsequently excited, the dye emits fluorescence proportional to theamount of double-stranded DNA (probe-target duplex) present. The sampleis then steadily heated while fluorescence is continually monitored. Arapid fall in fluorescence indicates the denaturing temperature of theprobe-target duplex. Using this technique, a single-base mismatchbetween the probe and target results in a significant lowering ofmelting temperature (T_(m)) that can be readily detected.

A variety of other techniques can be used to detect the polymorphisms inDNA. Merely by way of example, see U.S. Pat. Nos. 4,666,828; 4,801,531;5,110,920; 5,268,267; 5,387,506; 5,691,153; 5,698,339; 5,736,330;5,834,200; 5,922,542; and 5,998,137 for such methods.

Example 7 Expression of P-gp

The expression and purification of proteins, such as the P-gp, can beperformed using standard laboratory techniques. After expression,purified P-gp may be used for functional analyses, antibody production,diagnostics, and patient therapy. Furthermore, the DNA sequence of themdr1 cDNA can be manipulated in studies to understand the expression ofthe gene and the function of its product. Mutant forms of the caninemdr1 gene may be isolated based upon information contained herein, andmay be studied in order to detect alteration in expression patterns interms of relative quantities, tissue specificity, molecular weight,stability, and functional properties of the encoded mutant P-gp protein.Partial or full-length cDNA sequences, which encode for the subjectprotein, may be ligated into bacterial expression vectors. Methods forexpressing large amounts of protein from a cloned gene introduced intoEscherichia coli (E. coli) may be utilized for the purification,localization and functional analysis of proteins. For example, fusionproteins consisting of amino terminal peptides encoded by a portion ofthe E. coli lacZ or trpE gene linked to P-gp proteins may be used toprepare polyclonal and monoclonal antibodies against these proteins.Thereafter, these antibodies may be used to purify proteins byimmunoaffinity chromatography, in diagnostic assays to quantitate thelevels of protein and to localize proteins in tissues and individualcells by immunofluorescence.

Intact native protein may also be produced in E. coli in large amountsfor functional studies. Methods and plasmid vectors for producing fusionproteins and intact native proteins in bacteria are described inSambrook et al. (In Molecular Cloning: A Laboratory Manual, Ch. 17,CSHL, New York, 1989). Such fusion proteins may be made in largeamounts, are easy to purify, and can be used to elicit antibodyresponse. Native proteins can be produced in bacteria by placing astrong, regulated promoter and an efficient ribosome-binding siteupstream of the cloned gene. If low levels of protein are produced,additional steps may be taken to increase protein production; if highlevels of protein are produced, purification is relatively easy.Suitable methods are presented in Sambrook et al. (In Molecular Cloning:A Laboratory Manual, CSHL, New York, 1989) and are well known in theart. Often, proteins expressed at high levels are found in insolubleinclusion bodies. Methods for extracting proteins from these aggregatesare described by Sambrook et al. (In Molecular Cloning: A LaboratoryManual, Ch. 17, CSHL, New York, 1989). Vector systems suitable for theexpression of lacZ fusion genes include the pUR series of vectors(Ruther and Muller-Hill, EMBO J. 2:1791, 1983), pEX1-3 (Stanley andLuzio, EMBO J. 3:1429, 1984) and pMR100 (Gray et al., Proc. Natl. Acad.Sci. USA 79:6598, 1982). Vectors suitable for the production of intactnative proteins include pKC30 (Shimatake and Rosenberg, Nature 292:128,1981), pKK177-3 (Amann and Brosius, Gene 40:183, 1985) and pET-3(Studiar and Moffatt, J. Mol. Biol. 189:113, 1986). P-gp fusion proteinsmay be isolated from protein gels, lyophilized, ground into a powder andused as an antigen. The DNA sequence can also be transferred from itsexisting context to other cloning vehicles, such as other plasmids,bacteriophages, cosmids, animal viruses and yeast artificial chromosomes(YACs) (Burke et al., Science 236:806-812, 1987). These vectors may thenbe introduced into a variety of hosts including somatic cells, andsimple or complex organisms, such as bacteria, fungi (Timberlake andMarshall, Science 244:1313-1317, 1989), invertebrates, plants (Gasserand Fraley, Science 244:1293, 1989), and animals (Pursel et al., Science244:1281-1288, 1989), which cell or organisms are rendered transgenic bythe introduction of the heterologous mdr1 cDNA.

For expression in mammalian cells, the cDNA sequence may be ligated toheterologous promoters, such as the simian virus (SV) 40 promoter in thepSV2 vector (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076,1981), and introduced into cells, such as monkey COS-1 cells (Gluzman,Cell 23:175-182, 1981), to achieve transient or long-term expression.The stable integration of the chimeric gene construct may be maintainedin mammalian cells by biochemical selection, such as neomycin (Southernand Berg, J. Mol. Appl. Genet. 1:327-341, 1982) and mycophenolic acid(Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981).

DNA sequences can be manipulated with standard procedures such asrestriction enzyme digestion, fill-in with DNA polymerase, deletion byexonuclease, extension by terminal deoxynucleotide transferase, ligationof synthetic or cloned DNA sequences, site-directed sequence-alterationvia single-stranded bacteriophage intermediate or with the use ofspecific oligonucleotides in combination with PCR.

The cDNA sequence (or portions derived from it) or a mini gene (a cDNAwith an intron and its own promoter) may be introduced into eukaryoticexpression vectors by conventional techniques. These vectors aredesigned to permit the transcription of the cDNA in eukaryotic cells byproviding regulatory sequences that initiate and enhance thetranscription of the cDNA and ensure its proper splicing andpolyadenylation. Vectors containing the promoter and enhancer regions ofthe SV40 or long terminal repeat (LTR) of the Rous Sarcoma virus andpolyadenylation and splicing signal from SV40 are readily available(Mulligan et al., Proc. Natl. Acad. Sci. USA 78:1078-2076, 1981; Gormanet al., Proc. Natl. Acad. Sci. USA 78:6777-6781, 1982). The level ofexpression of the cDNA can be manipulated with this type of vector,either by using promoters that have different activities. For example,the baculovirus pAC373 can express cDNAs at high levels in S. frugiperdacells (Summers and Smith, In Genetically Altered Viruses and theEnvironment, Fields et al. (Eds.) 22:319-328, CSHL Press, Cold SpringHarbor, N.Y., 1985) or by using vectors that contain promoters amenableto modulation, for example, the glucocorticoid-responsive promoter fromthe mouse mammary tumor virus (Lee et al., Nature 294:228, 1982). Theexpression of the cDNA can be monitored in the recipient cells 24 to 72hours after introduction (transient expression).

In addition, some vectors contain selectable markers such as the gpt(Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981) orneo (Southern and Berg, J. Mol. Appl. Genet. 1:327-341, 1982) bacterialgenes. These selectable markers permit selection of transfected cellsthat exhibit stable, long-term expression of the vectors (and thereforethe cDNA). The vectors can be maintained in the cells as episomal,freely replicating entities by using regulatory elements of viruses suchas papilloma (Sarver et al., Mol. Cell. Biol. 1:486, 1981) orEpstein-Barr (Sugden et al., Mol. Cell. Biol. 5:410, 1985).Alternatively, one also can produce cell lines that have integrated thevector into genomic DNA. Both of these types of cell lines produce thegene product on a continuous basis. One can also produce cell lines thathave amplified the number of copies of the vector (and therefore of thecDNA as well) to create cell lines that can produce high levels of thegene product (Alt et al., J. Biol. Chem. 253:1357, 1978).

The transfer of DNA into eukaryotic, in particular human or othermammalian cells, is now a conventional technique. The vectors areintroduced into the recipient cells as pure DNA (transfection) by, forexample, precipitation with calcium phosphate (Graham and van der Eb,Virology 52:456-467, 1973) or strontium phosphate (Brash et al., Mol.Cell. Biol. 7:2013, 1987), electroporation (Neumann et al., EMBO J.1:841, 1982), lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA84:7413, 1987), DEAE dextran (McCuthan et al., J. Natl. Cancer Inst.41:351, 1968), microinjection (Mueller et al., Cell 15:579, 1978),protoplast fusion (Schafner, Proc. Natl. Acad. Sci. USA 77:2163-2167,1980), or pellet guns (Klein et al., Nature 327:70, 1987).Alternatively, the cDNA, or fragments thereof, can be introduced byinfection with virus vectors. Systems are developed that use, forexample, retroviruses (Bernstein et al., Gen. Engr. 7:235, 1985),adenoviruses (Ahmad et al., J. Virol. 57:267, 1986), or Herpes virus(Spaete et al., Cell 30:295, 1982). P-gp encoding sequences also can bedelivered to target cells in vitro via non-infectious systems, forinstance liposomes.

These eukaryotic expression systems can be used for studies of P-gpencoding nucleic acids and mutant forms of these molecules, the P-gpprotein and mutant forms of this protein.

Using the above techniques, the expression vectors containing the mdr1gene sequence or cDNA, or fragments or variants or mutants thereof, canbe introduced into canine cells, mammalian cells from other species, ornon-mammalian cells as desired. The choice of cell is determined by thepurpose of the introduction. For example, monkey COS cells (Gluzman,Cell 23:175-182, 1981) that produce high levels of the SV40 T antigenand permit the replication of vectors containing the SV40 origin ofreplication may be used. Similarly, Chinese hamster ovary (CHO), mouseNIH 3T3 fibroblasts or canine fibroblasts or lymphoblasts may be used.

This disclosure thus encompasses recombinant vectors that comprise allor part of the mdr1 gene or cDNA sequences, for expression in a suitablehost. The mdr1 DNA is operatively linked in the vector to an expressioncontrol sequence in the recombinant DNA molecule so that the P-gppolypeptide can be expressed. The expression control sequence may beselected from the group consisting of sequences that control theexpression of genes of prokaryotic or eukaryotic cells and their virusesand combinations thereof. The expression control sequence may bespecifically selected from the group consisting of the lac system, thetrp system, the tac system, the trc system, major operator and promoterregions of phage lambda, the control region of fd coat protein, theearly and late promoters of SV40, promoters derived from polyoma,adenovirus, retrovirus, baculovirus and simian virus, the promoter for3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, thepromoter of the yeast alpha-mating factors and combinations thereof.

The host cell, which may be transfected with a vector described herein,may be selected from the group consisting of E. coli, Pseudomonas,Bacillus subtilis, Bacillus stearothermophilus or other bacilli; otherbacteria; yeast; fungi; insect; mouse or other animal; or plant hosts;or human or canine tissue cells.

It is appreciated that, for mutant or variant mdr1 DNA sequences,similar systems are employed to express and produce the mutant product.In addition, fragments of the P-gp protein can be expressed essentiallyas detailed above. Such fragments include individual P-gp proteindomains or sub-domains, as well as shorter fragments such as peptides.P-gp protein fragments having therapeutic properties may be expressed inthis manner also.

Example 8 Production of P-gp Protein Specific Binding Agents

Monoclonal or polyclonal antibodies may be produced to either the normalP-gp or mutant forms (e.g., truncations) of this protein. Optimally,antibodies raised against these proteins or peptides would specificallydetect the protein or peptide with which the antibodies are generated.That is, an antibody generated to the P-gp or a fragment thereof wouldrecognize and bind the P-gp and would not substantially recognize orbind to other proteins found in human cells.

The determination that an antibody specifically detects P-gp is made byany one of a number of standard immunoassay methods; for instance, theWestern blotting technique (Sambrook et al., In Molecular Cloning: ALaboratory Manual, CSHL, New York, 1989). To determine that a givenantibody preparation (such as one produced in a mouse) specificallydetects P-gp by Western blotting, total cellular protein is extractedfrom human cells (for example, lymphocytes) and electrophoresed on asodium dodecyl sulfate-polyacrylamide gel. The proteins are thentransferred to a membrane (for example, nitrocellulose) by Westernblotting, and the antibody preparation is incubated with the membrane.After washing the membrane to remove non-specifically bound antibodies,the presence of specifically bound antibodies is detected by the use ofan anti-mouse antibody conjugated to an enzyme such as alkalinephosphatase. Application of an alkaline phosphatase substrate5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results inthe production of a dense blue compound by immunolocalized alkalinephosphatase. Antibodies that specifically detect P-gp will, by thistechnique, be shown to bind to P-gp band (which will be localized at agiven position on the gel determined by its molecular weight).Non-specific binding of the antibody to other proteins may occur and maybe detectable as a weak signal on the Western blot. The non-specificnature of this binding will be recognized by one skilled in the art bythe weak signal obtained on the Western blot relative to the strongprimary signal arising from the specific antibody-P-gp binding.

Substantially pure P-gp or protein fragments (peptides) suitable for useas an immunogen may be isolated from transfected or transformed cells asdescribed above. Concentration of protein or peptide in the finalpreparation is adjusted, for example, by concentration on an Amiconfilter device, to the level of a few micrograms per milliliter.Monoclonal or polyclonal antibody to the protein can then be preparedusing one of the following techniques.

A. Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes of P-gp, or specifically to thetruncation protein identified and isolated as described can be preparedfrom murine hybridomas according to the classical method of Kohler andMilstein (Nature 256:495-497, 1975) or derivative methods thereof.Briefly, a mouse is repetitively inoculated with a few micrograms of theselected protein over a period of a few weeks. The mouse is thensacrificed, and the antibody-producing cells of the spleen isolated. Thespleen cells are fused by means of polyethylene glycol with mousemyeloma cells, and the excess un-fused cells destroyed by growth of thesystem on selective media comprising aminopterin (HAT media). Thesuccessfully fused cells are diluted and aliquots of the dilution placedin wells of a microtiter plate where growth of the culture is continued.Antibody-producing clones are identified by detection of antibody in thesupernatant fluid of the wells by immunoassay procedures, such as ELISA,as originally described by Engvall (Meth. Enzymol. 70:419-439, 1980),and derivative methods thereof. Selected positive clones can be expandedand their monoclonal antibody product harvested for use. Detailedprocedures for monoclonal antibody production are described in Harlowand Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988).

B. Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogeneous epitopes ofa single protein can be prepared by immunizing suitable animals with theexpressed protein (Example 7), which can be unmodified or modified toenhance immunogenicity. Effective polyclonal antibody production isaffected by many factors related both to the antigen and the hostspecies. For example, small molecules tend to be less immunogenic thanothers and may require the use of carriers and adjuvant. Also, hostanimals vary in response to site of inoculations and dose, with eitherinadequate or excessive doses of antigen resulting in low titerantisera. Small doses (ng level) of antigen administered at multipleintradermal sites appear to be most reliable. An effective immunizationprotocol for rabbits can be found in Vaitukaitis et al. (J. Clin.Endocrinol. Metab. 33:988-991, 1971).

Booster injections can be given at regular intervals, and antiserumharvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony et al. (In Handbook of Experimental Immunology,Wier, D. (ed.) chapter 19. Blackwell, 1973). Plateau concentration ofantibody is usually in the range of about 0.1 to 0.2 mg/ml of serum(about 12 μM). Affinity of the antisera for the antigen is determined bypreparing competitive binding curves, as described, for example, byFisher (Manual of Clinical Immunology, Ch. 42, 1980).

C. Antibodies Raised Against Synthetic Peptides

A third approach to raising antibodies against P-gp or peptides is touse one or more synthetic peptides synthesized on a commerciallyavailable peptide synthesizer based upon the predicted amino acidsequence of P-gp or peptide. Polyclonal antibodies can be generated byinjecting these peptides into, for instance, rabbits.

D. Antibodies Raised by Injection of P-gp Encoding Sequence

Antibodies may be raised against P-gp proteins and peptides bysubcutaneous injection of a DNA vector that expresses the desiredprotein or peptide, or a fragment thereof, into laboratory animals, suchas mice. Delivery of the recombinant vector into the animals may beachieved using a hand-held form of the Biolistic system (Sanford et al.,Particulate Sci. Technol. 5:27-37, 1987) as described by Tang et al.(Nature 356:152-154, 1992). Expression vectors suitable for this purposemay include those that express the P-gp encoding sequence (mdr1 gene orcDNA, for instance) under the transcriptional control of either thehuman β-actin promoter or the cytomegalovirus (CMV) promoter.

Antibody preparations prepared according to these protocols are usefulin quantitative immunoassays which determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively to identify the presence of antigenin a biological sample; or for immunolocalization of P-gp.

For administration to specific animal subjects (such as human or canineindividuals), antibodies, e.g., P-gp specific monoclonal antibodies, canbe adapted to be more effective in the target organism by methods knownin the art. By way of example, antibodies with a desired bindingspecificity can be commercially humanized (Scotgene, Scotland, UK;Oxford Molecular, Palo Alto, Calif.).

Example 9 Protein-Based Detection

An alternative method of detecting sensitivity to ivermectin and relateddrugs (those that are kept from crossing the blood-brain barrier byP-gp) is to examine the level or molecular weight (apparent size, e.g.,on after SDS-PAGE with or without immunodetection) of P-gp in the cellsof an individual. These diagnostic tools would be useful for detectingreduced levels of P-gp that result from, for example, mutations in thepromoter regions of the mdr1 gene or mutations within the coding regionof the gene that produced truncated, non-functional or unstablepolypeptides, as well as from deletions of a portion of or the entiremdr1 gene.

Localization and/or coordinated mdr1 expression (temporally orspatially) can also be examined using known techniques, such asisolation and comparison of P-gp from cell or tissue specific, or timespecific, samples. The determination of reduced or increased P-gplevels, in comparison to such expression in a control cell (e.g.,normal, as in taken from an individual not exhibiting sensitivity toivermectin or another neurotoxin), would be an alternative orsupplemental approach to the direct determination of mdr1 gene mutation(e.g., truncation mutation) status by the methods outlined above andequivalents.

The availability of antibodies specific to P-gp will facilitate thedetection, measurement (e.g., molecular weight determination) andquantitation of cellular P-gp by one of a number of immunoassay methodswhich are well known in the art and are presented in Harlow and Lane(Antibodies, A Laboratory Manual, CSHL, New York, 1988). Methods ofconstructing such antibodies are discussed above, in Example 8.

Any standard immunoassay format (e.g., ELISA, Western blot, or RIAassay) can be used to measure P-gp polypeptide or protein levels;comparison is to wild-type (normal) P-gp, and an alteration in P-gppolypeptide may be indicative of an abnormal biological conditionregarding resistance to potential neurotoxins, such as ivermectin.Immunohistochemical techniques may also be utilized for P-gp polypeptideor protein detection. For example, a tissue sample may be obtained froma subject, and a section stained for the presence of P-gp using a P-gpspecific binding agent (e.g., anti-P-gp antibody) and any standarddetection system (e.g., one which includes a secondary antibodyconjugated to horseradish peroxidase). General guidance regarding suchtechniques can be found in, e.g., Bancroft and Stevens (Theory andPractice of Histological Techniques, Churchill Livingstone, 1982) andAusubel et al. (Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1998).

For the purposes of quantifying or determining the estimated molecularweight of P-gp, a biological sample of the subject (which can be anyanimal, for instance a dog or a human), which sample includes cellularproteins, is required. Such a biological sample may be obtained frombody cells, such as those present in peripheral blood, urine, saliva,tissue biopsy, amniocentesis samples, surgical specimens and autopsymaterial. Quantitation and/or measurement of P-gp can be achieved byimmunoassay and compared to level and apparent size of the protein foundin control cells (e.g., healthy, as in from an individual known not tohave ivermectin sensitivity). A significant (e.g., 10% or greater)reduction in the amount of P-gp in the cells of a test subject comparedto the amount of P-gp found in normal cells, or a substantial reductionin the apparent molecular weight of the P-gp (e.g., as would be apparentwith a truncation mutation) could be taken as an indication that thesubject may have deletions or mutations in the mdr1 gene. Deletionand/or mutation of or within the mdr1-encoding sequence, and substantialunder-expression of P-gp, may be indicative of altered sensitivity toivermectin and other drugs that are usually kept from crossing theblood-brain barrier by P-gp.

Merely by way of example, canine P-gp can be analyzed as described inMealey et al., Cancer Letters 126:187-192, 1998. For instance,immunoblotting has been carried out using the following procedure:

Cells were harvested by trypsinization, washed with DPBS and solubilizedin tumor solubilization buffer (TSB, 50 mM Tris-HCl (pH 6.8), 50 mM KCl,5 mM EGTA, 5 mM MgCl₂, 2% CHAPS, 0.1 mM leupeptin, 0.2 mMphenylmethyl-sulfonylfluoride and 10 mM dithiothreitol). Insolublecomplexes were cleared by a 5-minute spin (1500 revolutions/minute) andthe soluble protein was collected for quantitation using a modifiedLowry technique (Lowry, J. Biol. Chem. 193:265-275, 1951).

Protein samples were separated by SDS-PAGE and electroblotted onto anImmobilon-P™ membrane (Millipore, Bedford, Mass.). Membranes were washedwith blot to buffer (50 mM Tris-HCl, 2 mM CaCl₂, 80 mM NaCl, 5% non-fatdry milk, 0.2% Nonidet P-40 and 0.03% sodium azide) for one hour at 25°C. and then incubated (25° C. for 16 hours) with a murine anti-humanP-glycoprotein monoclonal antibody (C219; Signet, Dedham, Mass.). Actinwas subsequently detected using a monoclonal anti-actin antibody (ICNImmunobiologicals, Costa Mesa, Calif.). Membranes were washed in freshblotto (non-fat milk) buffer and incubated with the appropriate alkalinephosphatase-labeled secondary antibody. Membranes were washed withbuffer A (50 mM Tris-HCl, 2 mM CaCl₂ and 80 mM NaCl) and developed usingnitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate with analkaline-phosphatase conjugate substrate kit (BioRad, Hercules, Calif.).The color reaction was terminated by washing in distilled water. Theresulting bands were scanned with a Visage 110 camera-based densitometer(Bio-Image, Ann Arbor, Mich.) and analyzed using whole band software.Integrated intensity signals for P-glycoprotein can be normalized tothose of a protein the level of which is not expected to change underthe experimental conditions.

Example 10 Differentiation of Individuals Homozygous Versus Heterozygousfor the Polymorphism(s)

As will be appreciated, the oligonucleotide ligation assay (OLA), asdescribed at Nickerson et al. (Proc. Natl. Acad. Sci. USA 87:8923-8927,1990), allows the differentiation between individuals who are homozygousversus heterozygous for the herein-described frame-shift truncationmutation in mdr1. This feature allows one to rapidly and easilydetermine whether an individual is homozygous for at least a neurotoxinsensitivity-linked polymorphism, which condition can result inneurotoxicosis and possible death when an individual is administered anotherwise safe drug dosage. Alternatively, OLA can be used to determinewhether a subject is homozygous for either of these polymorphisms.

As an example of the OLA assay, when carried out in microtiter plates,one well is used for the determination of the presence of the mdr1allele that contains the herein-described frame-shift truncationmutation, and a second well is used for the determination of thepresence of the mdr1 wild-type allele. Thus, the results for anindividual who is heterozygous for the polymorphism will show a signalin each of the “truncated” and wild-type wells, and an individual who ishomozygous for one allele or the other will show a signal only in thecorresponding well.

Likewise, truncation itself can be used to detect heterogeneity in theP-gp protein. Because truncation leads to production of a shorter (andtherefore “lighter”) protein product, Western analysis can be used todistinguish between heterozygous and homozygous individuals, as well asbetween homozygous truncated (and therefore sensitive) and wild-type(resistant) individuals.

Example 11 Kits

Kits are provided which contain the necessary reagents for determiningthe presence or absence of polymorphism(s) in a P-gp-encoding sequence,such as probes or primers specific for the mdr1 gene. Such kits can beused with the methods described herein to determine whether anindividual is likely to be sensitive to ivermectin and other drugs thatare usually kept from crossing the blood-brain barrier by P-gp (such asthose listed in Table II).

The provided kits may also include written instructions. Theinstructions can provide calibration curves or charts to compare withthe determined (e.g., experimentally measured) values.

Kits are also provided to determine altered (e.g., lowered) expressionof mRNA (i.e., containing probes) or P-gp protein (i.e., containingantibodies or other P-gp-specific binding agents), as well as truncatedP-gp.

A. Kits for Detecting mdr1 Nucleic Acid Mutations

The oligonucleotide probes and primers disclosed herein can be suppliedin the form of a kit, for use in detection of ivermectin sensitivity ina subject. In such a kit, an appropriate amount of one or more of theoligonucleotides is provided in one or more containers. Theoligonucleotides may be provided suspended in an aqueous or othersolution, or as a freeze-dried or lyophilized powder, for instance. Thecontainer(s) in which the oligonucleotide(s) are supplied can be anyconventional container that is capable of holding the supplied form, forinstance, microfuge tubes, ampoules, or bottles. In some applications,pairs of primers may be provided in pre-measured single use amounts inindividual, typically disposable, tubes or equivalent containers. Withsuch an arrangement, the sample to be tested for the presence of an mdr1polymorphism can be added to the individual tubes and amplification orother laboratory manipulation carried out directly.

The amount of each oligonucleotide supplied in the kit can be anyappropriate amount, depending for instance on the market to which theproduct is directed. For instance, if the kit is adapted for research orclinical use, the amount of each oligonucleotide primer provided wouldlikely be an amount sufficient to prime several in vitro nucleic acidamplification reactions. Those of ordinary skill in the art know theamount of oligonucleotide primer that is appropriate for use in a singleamplification reaction. General guidelines may for instance be found inInnis et al. (PCR Protocols, A Guide to Methods and Applications,Academic Press, Inc., San Diego, Calif., 1990), Sambrook et al. (InMolecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989),and Ausubel et al. (In Current Protocols in Molecular Biology, GreenePubl. Assoc. and Wiley-Intersciences, 1992).

A kit may include more than two primers, in order to facilitate the invitro amplification of mdr1 sequences, for instance the mdr1 gene or the5′- or 3′-flanking region thereof.

In some embodiments, kits may also include the reagents necessary tocarry out nucleotide amplification reactions, including, for instance,DNA sample-preparation reagents, appropriate buffers (e.g., polymerasebuffer), salts (e.g., magnesium chloride), and deoxyribonucleotides(dNTPs).

Kits may include either labeled or unlabeled oligonucleotide probes foruse in detection of mdr1 polymorphism(s). In certain embodiments, theseprobes will be specific for a potential polymorphism that may be presentin the target sequence. The appropriate sequences for such a probe willbe any sequence that includes all or part of the identified polymorphicsite, particularly nucleotide positions 294 through 297 of the caninemdr1 gene, such that the sequence the probe is complementary to thetruncation polymorphic site and a portion of the surrounding mdr1sequence. An oligonucleotide including the sequenceAAACATGAAGATAGCTTTGCAAAT (corresponding to residues 284-309 of SEQ IDNO: 1) exemplifies such a sequence, and a probe useful for disclosedmethods could comprise this sequence. Alternatively, an example of aprobe specific for detecting the ivermectin sensitivity allele of mdr1may include the following sequence: AAACATGACAGCTTTGCAAAT (alsocorresponding to residues 284-309 of SEQ ID NO: 1, but with residues294-297 removed).

It also may be advantageous to provide in the kit one or more controlsequences for use in the amplification reactions. The design ofappropriate positive and negative control sequences is well known to oneof ordinary skill in the appropriate art.

B. Kits for Detection of mdr1 mRNA

Kits similar to those disclosed above for the detection of mdr1polymorphisms directly can be used to detect mdr1 mRNA expression, suchas over- or under-expression or expression of a truncated form. Suchkits include an appropriate amount of one or more oligonucleotideprimers for use in, for instance, reverse transcription nucleic acidamplification reactions (e.g., RT-PCR), similarly to thoseoligonucleotides described above with art-obvious modifications for usewith RNA amplification.

In some embodiments, kits for detection of altered expression of mdr1mRNA may also include some or all of the reagents necessary to carry outRT-PCR or other in vitro amplification reactions, for instance, RNAsample preparation reagents (including e.g., an RNase inhibitor),appropriate buffers (e.g., polymerase buffer), salts (e.g., magnesiumchloride), and deoxyribonucleotides (dNTPs). Written instructions alsomay be included.

Such kits may in addition include either labeled or unlabeledoligonucleotide probe(s) for use in detection of the in vitro amplifiedtarget sequences. The appropriate sequences for such a probe will be anysequence that falls between the annealing sites of the two providedoligonucleotide primers, such that the sequence the probe iscomplementary to is amplified during the in vitro amplificationreaction. In certain embodiments, these probes will be specific for apotential polymorphism that may be present in the amplified targetsequences.

It also may be advantageous to provide in the kit one or more controlsequences for use in the RT-PCR reactions. The design of appropriatepositive and negative control sequences is well known to one of ordinaryskill in the appropriate art.

Alternatively, kits may be provided with the necessary reagents to carryout quantitative or semi-quantitative Northern analysis of mdr1 mRNA.Such kits include, for instance, at least one mdr1-specificoligonucleotide for use as a probe. This oligonucleotide may be labeledin any conventional way, including with a selected radioactive isotope,enzyme substrate, co-factor, ligand, chemiluminescent or fluorescentagent, hapten, or enzyme. In certain embodiments, such probes will bespecific for a potential polymorphism (e.g., a truncation mutation) thatmay be present in the target sequences.

C. Kits For Detection of P-gp Expression

Kits for the detection of P-gp protein expression (such as over- orunder-expression), and particularly changes in the apparent molecularweight of expressed P-gp, are also encompassed herein. Such kits mayinclude at least one target-protein-specific binding agent (e.g., apolyclonal or monoclonal antibody or antibody fragment that specificallyrecognizes P-gp) and may include at least one control (such as adetermined amount of P-gp, with a defined molecular weight or apparentsize, or a sample containing a determined amount of P-gp). TheP-gp-protein-specific binding agent and control may be contained inseparate containers.

P-gp expression detection kits may also include a means for detectingP-gp:binding agent complexes, for instance the agent may be detectablylabeled. If the binding agent is not labeled, it may be detected bysecond antibodies or protein A for example, which components may also beprovided in some kits in one or more separate containers. Such detectiontechniques are known.

Additional components in specific kits may include instructions forcarrying out the assay. Instructions will allow the tester to determinewhether P-gp expression levels are elevated, and/or whether theexpressed P-gp has an altered molecular weight compared to wild-typeP-gp. Reaction vessels and auxiliary reagents such as chromogens,buffers, enzymes, etc. may also be included in the kits.

D. Kits for Detection of Homozygous versus Heterozygous Allelism

Also provided are kits that allow differentiation between individualswho are homozygous versus heterozygous for a truncation mutation in themdr1 gene. Certain examples of such kits provide the materials forperforming oligonucleotide ligation assays (OLA), as described byNickerson et al., Proc. Natl. Acad. Sci. USA 87:8923-8927, 1990. Inspecific embodiments, these kits contain one or more microtiter plateassays, designed to detect polymorphism(s) in the mdr1 sequence of asubject, as described herein.

In other examples of such kits, materials are provided for examining thesize of the P-gp expressed by an individual, for instance components forcarrying out a Western analysis or other immunological assay.

Additional components in some of these kits may include instructions forcarrying out the assay. Instructions will allow the tester to determinewhether an mdr1 truncation mutation allele is homozygous orheterozygous, either through examination of nucleic acid molecules orprotein. Reaction vessels and auxiliary reagents such as chromogens,buffers, enzymes, etc. may also be included in the kits.

It may also be advantageous to provide in the kit one or more controlsequences for use in the OLA or immunoassay reactions. The design ofappropriate positive and negative control molecules is well known to oneof ordinary skill in the appropriate art.

Example 12 Animal Model

A large number of P-gp substrates are routinely prescribed in humans,including HIV-1 protease inhibitors and many chemotherapeutic agents.Animals (e.g., Collies) possessing a polymorphism of the mdr1 gene, forinstance, the truncation mutation described herein, can serve as auseful model for studying the effects of P-glycoprotein substrates onhumans with mdr1 polymorphisms. In addition, these animals can serve asmodels for studying the effects of compounds that interact with P-gp.They are also useful to study pharmacologic inhibition of P-gp, forinstance in order to identify or characterize modulators of P-gptransport activity that may be useful to increase (or decrease, orregulate) drug absorption in or distribution to one or more tissues in asubject.

Use of the animals identified herein (particularly Collies) possessing anaturally-occurring polymorphism of the mdr1 gene avoids confoundingeffects attributable to producing mdr1 mutations through geneticengineering, and is expected to result in better acceptance as aresearch model by both the research community and society.

Using methods described herein, individual animals are identified asbeing heterozygous or homozygous for an mdr1 truncation, and theseanimals are used as subjects for animal model studies. In specificexamples, a drug of interest (or drug candidate or other compound ormixture thereof) is administered to Collies possessing a polymorphism ofMDR-1, and the effects are monitored systemically, for instance, or inparticular tissues. Routes of administration include but are not limitedto oral and parenteral routes, such as intravenous (iv), intraperitoneal(ip), rectal, topical, ophthalmic, nasal, and transdermal.

Effective doses of the compound(s) of interest can be determined by oneof ordinary skill in the art, and may be tailored to the specificexperiments being run. In some embodiments, the compound is administeredwith a goal of achieving tissue concentrations that are at least as highas the IC₅₀ of the compounds(s) tested. An example of such a dosagerange is 0.1 to 200 mg/kg body weight. The specific dose level andfrequency of dosage for any particular subject may be varied and willdepend upon a variety of factors, including the activity of the specificcompound, the metabolic stability and length of action of that compound,the age, body weight, mode and time of administration, and the rate ofexcretion of the compound.

Systemic effects of the compound of interest can be monitored in theanimal following its administration. For example, neurological symptomslinked to a drug passing across one or more cellular membranes via P-gpcan include salivation, vomiting, confusion, ataxia, tremors,seizure-like activity, recumbency, non-responsiveness, and coma.Alternatively, drug or other compound levels in the blood, orbiochemical changes in the gastrointestinal tract, kidneys, nervoussystem, liver, tumor cells, or other tissues, can be monitored.

The animal model can also be used to identify and/or characterize testcompounds known to, or expected to, modulate the activity of P-gp. Inexamples of such methods, the animals are treated (e.g., concurrently orsequentially) with both the test compound and a compound known to betransported by P-gp (the effector molecule). The animals are thenmonitored to detect one or more effects caused by the effector compound,and the level of effect can be compared to animals that were not treatedwith the test compound (or which received a different treatmentregimen). Depending on the effector molecule used, different biologicalcharacteristics of the animal, or a tissue or cell within the animal,can be monitored to determine whether (and to what extent) the testcompound influences P-gp interaction with the effector. Depending onwhether the test molecule increases or decreases P-gp transport of theeffector, the test molecule is identified as an agonist or antagonist ofP-gp activity and can be selected for further characterization.

Example 13 Cultured Cells

Cells or tissues from animals (e.g., Collies) possessing a polymorphismof the mdr1 gene, for instance, the truncation mutation describedherein, also can serve as useful model systems for studying the effectsof P-gp interacting molecules. In addition, cells and tissues fromanimals possessing a polymorphism of the mdr1 gene can serve as modelsfor studying pharmacologic inhibition, stimulation, and/or regulation ofP-gp.

In some examples of such cell-based models, cells from an animalcarrying an mdr1 truncation as described herein are transformed (stablyor transiently) with an mdr1 construct, for instance which comprisesknown (e.g., engineered) or unknown mutations (e.g., point mutationssuch as a naturally occurring polymorphism, for instance polymorphismsidentified in human mdr1). Such cellular expression systems enableexamination of the activity of the mutant mdr1 construct in a definedlarge mammal background.

P-gp is expressed in many tissues throughout the mammalian body,including the epithelium of the gastrointestinal tract, renal-tubularepithelium, brain capillary endothelial cells, biliary tubularepithelial cells, and at the plasma membrane in many tumor cells. Insome examples, cells used for the in vitro cell system are selected froma specific tissue or cell type, so that tissue or cell specific effectsof P-gp transport can be studied. In carrying out such methods, specifictissues are isolated from canines (e.g., Collies) possessing apolymorphism of the mdr1 gene, such as the truncation described herein.Though methods are widely known for isolating cells and tissues ofspecific types, the following procedures are provided as specificexamples and can be used to isolate specific cells from canine samples:

-   -   (a) Canine intestinal epithelial cells can be isolated and        cultured as described in Koop and Buchan, Gastroenterology 102:        28-34, 1992.    -   (b) Canine brain microvessel endothelial cells can be isolated        and cultured as described in Drewes et al., Brain Research        Bulletin 21: 771-776, 1988.    -   (c) Canine renal-tubular cells can be isolated and cultured as        described in Hamada et al., Nephron. 68: 104-111, 1994.    -   (d) Canine hepatocytes can be isolated and cultured as described        in Lu and Li, Chem. Biol. Interact. 143: 271-281, 2001, Amacher        and Martin, Fundam. Appl Toxicol. 40:256-263, 1997, or Placidi        et al., Drug Metab. Dispos. 25: 94-99, 1997.    -   (e) Neoplasms can be induced using techniques well known in the        art, including but not limited to the use of chemical        carcinogens, viruses, and radiation exposure. Alternatively,        naturally occurring tumors can also serve as a source for        cancerous cells. Tumor cells can then be cultured as described        in Lehr et al., Anticancer Res. 18(6A): 4483-4488, 1998.

The cell-based model can also be used to identify and/or characterizetest compounds known to, or expected to, modulate the activity of P-gp.In examples of such methods, the cells are contacted (e.g., concurrentlyor sequentially) with both the test compound and a compound known to betransported by P-gp (the effector molecule). The cells are thenmonitored to detect one or more effects caused by the effector compound,and the level of effect can be compared to animals that were not treatedwith the test compound (or which received a different treatmentregimen). Depending on the effector molecule used, different biologicalcharacteristics of the cells can be monitored to determine whether (andto what extent) the test compound influences P-gp interaction with theeffector. Depending on whether the test molecule increases or decreasesP-gp transport of the effector, the test molecule is identified as anagonist or antagonist of P-gp activity and can be selected for furthercharacterization.

Compound transport across a membrane in cultured cells can be monitoredusing known techniques, for instance the radio-labeling method outlinedin Schinkel et al. J. Clin. Invest. 96:1698-1705, 1995. Briefly, cellsare grown in complete medium including L-glutamine, penicillin,streptomycin, and FCS and seeded on microporous polycarbonate membranefilters (3.0 μM pore size, 24.5 mm diameter, Transwell™ 3414, CostarCorp., Cambridge, Md.) at a density of 2×10⁶ cells per well. Medium ateither the apical or basal side of the cell layer is replaced withcomplete medium containing the appropriate concentration of radiolabeleddrug or drug candidate and cells are incubated at 37° C. in 5% CO₂.Subsequently, 50 μl aliquots are taken from each compartment at varioustime intervals, for instance 1, 2, 3, and 4 hours. The appearance ofradioactivity in the non-treated compartment is measured and presentedas the fraction of total radioactivity added at the beginning of theexperiment.

In some methods employing the provided cell systems, the level ofradioactive effector compound transported into the cells is comparedbetween cells that received treatment with a test compound (e.g., aputative or potential P-gp activity agonist or antagonist) and thosethat did not receive such treatment, or between cells that receiveddifferent test compound treatments (for instance, as to the testcompound used, the method or timing of application, the amount applied,and so forth).

Methods of monitoring compound-uptake will in some instances beinfluenced by the compounds being studied.

This disclosure provides a mutation in the canine (particularly collie)mdr1 gene, which results in a truncation of P-gp and leads to extremesensitivity to ivermectin in animals homozygous for this mutant allele.The disclosure further provides methods and kits for screening for thismutation, in order to identify animals susceptible to toxicity fromivermectin and other neurotoxic compounds that interact with the P-gptransport protein. Also provided are whole-animal and cell-based modelsystems for studying compound interactions with P-gp using the mdr1truncation mutation described herein. It will be apparent that theprecise details of the methods described may be varied or modifiedwithout departing from the spirit of the described invention. We claimall such modifications and variations that fall within the scope andspirit of the claims below.

1. A method of screening for whether a compound has a biological effectin a canine cell having a truncated P-gp protein, comprising: contactinga first canine cell with the compound, wherein the first canine cell hasa truncation mutation in a mdr1 gene at residues 294-297 of SEQ ID NO:1, which mutation results in the canine cell having a truncated P-gpprotein, to produce a contacted canine cell; and comparing acharacteristic of the contacted canine cell with a) the samecharacteristic of the first canine cell prior to contact with thecompound, b) the same characteristic of a similar canine cell notcontacted with the compound, or c) the same characteristic of a secondcanine cell contacted with the compound, wherein the second canine celldoes not have a truncation mutation in its mdr1 gene; wherein adifference in the characteristic indicates the compound has a biologicaleffect in the canine cell having the truncated P-gp protein.
 2. Themethod of claim 1, wherein contacting the canine cells with the compoundoccurs in vivo.
 3. The method of claim 1, wherein the canine cell is agastrointestinal tissue cell, a renal tissue cell, a brain capillaryendothelial cell, a liver tissue cell, a placental cell, bronchiolarepithelial cell, or an adrenal cortical cell.
 4. The method of claim 1,wherein the characteristic of the cell being compared is genetic,physiological, chemical, or morphological.
 5. The method of claim 1,wherein the biological effect results from a defect in efflux of thecompound from the contacted canine cell.
 6. The method of claim 1,wherein the first canine cell is a neoplastic canine cell.
 7. The methodof claim 1, wherein the compound is a neurokinin receptor antagonist,anti-emetic agent, beta-adrenergic receptor antagonist, antiinfectiveagent, antiepileptic agent, antineoplastic agent, analgesic agent,anti-psychotic agent, anti-parasitic agent or anti-depressive agent. 8.The method of claim 1, wherein the compound is an antiviral agent. 9.The method of claim 1, wherein the canine cell is a dog cell.
 10. Themethod of claim 9, wherein the dog cell is a Collie cell.
 11. The methodof claim 1, wherein the first canine cell is contacted in vitro.
 12. Themethod of claim 2, wherein contacting the first canine cell in vivocomprises administering the compound to a dog.